This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Debranching enzyme, Dbr1p, is an RNase that promotes intron RNA turnover after splicing. The enzyme is a phosphoesterase that cuts the 2'-5'bond at intron RNA lariat branch points, allowing exonucleases to access the RNA. Loss of Dbr1p function results in accumulation of intron lariat RNAs, which has a minimal effect on yeast cell viability. A second phenotype associated with loss of Dbr1p function in yeast is decreased replication of the retrovirus-like elements Ty1 and Ty3, with reverse transcription being the specific replication stage affected. This latter phenotype is not understood, however a group in California has shown that the retrovirus HIV-1 requires the human version of the Dbr1 enzyme for reverse transcription. A goal of the Menees research group has been to understand how Dbr1p acts as a host factor for retroviruses and retrovirus-like elements. One approach for achieving this goal is to determine the interacting partners for yeast Dbr1p. A yeast strain containing a tandem affinity tagged (TAP-tagged) DBR1 allele as its only source of Dbr1p was used to purify Dbr1p-TAP. This yeast strain is wild-type for the processes that depend on Dbr1p: RNA lariat debranching and Ty element transposition. Mass spectrometric analysis revealed that Ty1 proteins were found to specifically copurify with Dbr1p-TAP, raising the possibility that Dbr1p is present within Ty1 virus-like particles (VLPs). Such an association would support a specific host factor role for Dbr1p in Ty1 replication. However, contrasting data were obtained with partially purified Ty1 VLP preparations: Dbr1p was detected by western blotting but not by mass spectrometry. Using more purified Ty1 VLPs obtained by an immunoaffinity method, Dbr1p is still not detected by mass spectrometry. We are distinguishing between the following two possibilities: (1) Dbr1p may be present in VLPs but mass spectrometry may not be able to reveal its presence or (2) the TAP-tagged version of Dbr1p spuriously associates with VLPs. The development of an immunoaffinity method to purify Ty1 VLPs opens up a new window for studying Ty1 host factors. Such studies will be helpful in assessing the roles of host factors for human retroviruses.